The present invention provides novel enhancers of net photosynthesis and methods of enhancing net photosynthesis and plant growth. Due to the ever increasing need to grow crops more efficiently, the ability to increase a plant's growth rate would be of great social and economic value. An attractive biochemical target to accomplish this is the plant's ability to fix carbon dioxide by photosynthesis. The ability to enhance net photosynthesis will allow the grower to generate a plant that is faster growing and/or has increased biomass in, for example, storage products, such as starch (polysaccharides), protein or fats.
Researchers have been investigating means to boost a plant's photosynthetic rate in a variety of ways. Some examples include exposing a plant to light with the same wavelengths as sunlight (see, e.g., JP04166017); generating transgenic plants with exogenous genes involved in photosynthesis (see, e.g., WO 96/21737); application of various chemicals to increase the rate of photosynthesis (see, e.g., JP03109304, U.S. Pat. Nos. 4,704,161 and 5,597,400); and applying chemicals to suppress photorespiration, resulting in an increase in the efficiency of photosynthesis (see, e.g., JP01086822, JP60115501, JP55136205, JP55036437).
However, prior to this invention, no enhancers of net photosynthesis have been identified. Furthermore, the present invention taps a rich, yet to date untapped source, of enhancers of net photosynthesis from microorganisms, particularly bacteria. Identification of enhancers of net photosynthesis and plant growth from microorganisms would offer many benefits. For example, the reagent, as a natural product, can be generated on a large, industrial scale using conventional techniques. This eliminates the need to structurally analyze and/or synthetically produce the bioactive component of the microbial extract. Furthermore, if the microorganism can grow in soil or can colonize plant roots, application of the microbe to the soil could be equivalent to applying the bacterial natural product or extract itself. If the genetic mechanism responsible for producing the bioactive agent is found, transgenic microorganisms can be constructed. Thus, any soil-borne or plant (e.g., root, shoot, leaf)-colonizing microorganism can be recombinantly manipulated to generate an enhancer of net photosynthesis or enhancer of plant growth.
Taxonomic labels on the most frequently used rhizobia have been in a state of flux for the past decade, but the two most important species currently are known as Sinorhizobium meliloti for alfalfa and Bradyrhizobium japonicum for soybean (Phillips and Martinez-Romero, 2000, Biological nitrogen fixation. In: Encyclopedia of Microbiology, 2nd edition, ed. J. Lederberg. Academic Press, San Diego. Vol 1: In press.) A less studied species, Sinorhizobium fredii, formerly called Rhizobium fredii, forms root nodules on both alfalfa and soybean (Hashem et al, 1997, Symbiosis 22:255-264) and thus may have outstanding commercial potential for future inoculants.
Genetic attempts to improve the competitiveness and commercial performance of rhizobia have centered on a small number of traits, including enhancement of N2-fixation capacity (Bosworth et al, 1994, Appl. Environ. Microbiol. 60:3815-3832), production of antibiotics that impair competing bacteria (Kent et al, 1998, Appl. Environ. Microbiol. 64:1657-1662), and the use of H2 as an energy source (Kent et al, 1998, Appl. Environ. Microbiol. 64:1657-1662). Other plant-associated bacteria are claimed to promote plant growth by supplying plant hormones, rather than by reducing N2 to ammonia. Azospirillum, one genus representative of this group, has been used successfully as an agricultural inoculant throughout the world for more than 25 years (Okon and Labandera-Gonzalez, 1994, Soil Biol. Biochem. 26:1591-1601). Although it is known from the literature that riboflavin synthesis can limit N2-fixation capacity in rhizobia (Pankthurst et al, 1974, Plant Physiol. 53:198-205), there are no published reports on increasing the production of this compound in Rhizobiaceae bacteria. Indeed, no genes in the Rhizobiaceae have been identified as contributing to riboflavin synthesis.
The biosynthetic pathway for riboflavin has been defined (Young, 1986, Nat. Prod. Rpt. 3:395-419), and genes associated with various steps in the pathway have been characterized in Escherichia coli (Eberhardt et al, 1996, Eur. J. Biochem. 242: 712-719; Richter et al, 1992, J. Bact. 174:4050-4056; Richter et al, 1997, J. Bact. 179:2022-2028), Bacillus subtilis (Coquard et al, 1997, Mol. Gen. Genet. 254:81-84; Perkins and Pero, 1993, In: Bacillus subtilis and Other Gram-Positive Bacteria, A. L. Sonenshein et al eds. Amer. Soc. Microbiol. Washington, D.C. p. 319-325; Schott et al, 1990, J. Biol. Chem. 265: 4204-4209), and several other bacteria outside the Rhizobiaceae. Commercial production of riboflavin was facilitated by expressing multiple copies of the rib operon in B. subtilis to achieve riboflavin titers of 4.5 g/liter (Stepanov et al, 1984, French patent application 3599355).
The present invention, by providing novel enhancers of net photosynthesis and enhancers of plant growth, particularly enhancers that can be synthesized by microbes and isolated from microbial extracts, fulfills these, and other, needs.